Phosphatidylinositol 3-kinase (PI3K) has been shown to enhance native voltage-dependent calcium channel (Ca(v)) currents both in myocytes and in neurons; however, the mechanism(s) responsible for this regulation were not known. Here we show that PI3K promotes the translocation of GFP-tagged Ca(v) channels to the plasma membrane in both COS-7 cells and neurons. We show that the effect of PI3K is mediated by Akt/PKB and specifically requires Ca(v)beta(2) subunits. The mutations S574A and S574E in Ca(v)beta(2a) prevented and mimicked, respectively, the effect of PI3K/Akt-PKB, indicating that phosphorylation of Ser574 on Ca(v)beta(2a) is necessary and sufficient to promote Ca(v) channel trafficking.

We have previously reported that, in venous myocytes, Gbetagamma scavengers inhibit angiotensin AT1A receptor-induced stimulation of L-type Ca2+ channels (1). Here, we demonstrate that intracellular infusion of purified Gbetagamma complexes stimulates the L-type Ca2+ channel current in a concentration-dependent manner. Additional intracellular dialysis of GDP-bound inactive Galphao or of a peptide corresponding to the Gbetagamma binding region of the beta-adrenergic receptor kinase completely inhibited the Gbetagamma-induced stimulation of Ca2+ channel currents. The gating properties of the channel were not affected by intracellular application of Gbetagamma, suggesting that Gbetagamma increased the whole-cell calcium conductance. In addition, both the angiotensin AT1A receptor- and the Gbetagamma-induced stimulation of L-type Ca2+ channels were blocked by pretreatment of the cells with wortmannin, at nanomolar concentrations. Correspondingly, intracellular infusion of an enzymatically active purified recombinant Gbetagamma-sensitive phosphoinositide 3-kinase, PI3Kgamma, mimicked Gbetagamma-induced stimulation of Ca2+ channels. Both Gbetagamma- and PI3Kgamma-induced stimulations of Ca2+ channel currents were reduced by protein kinase C inhibitors suggesting that the Gbetagamma/PI3Kgamma-activated transduction pathway involves a protein kinase C. These results indicate for the first time that Gbetagamma dimers stimulate the vascular L-type Ca2+ channels through a Gbetagamma-sensitive PI3K.

contributed equally to this workWe have cloned and characterized a new member of the voltage-dependent Ca 2+ channel g subunit family, with a novel gene structure and striking properties. Unlike the genes of other potential g subunits identi®ed by their homology to the stargazin gene, CACNG7 is a ®ve-, and not four-exon gene whose mRNA encodes a protein we have designated g 7 . Expression of human g 7 has been localized speci®cally to brain. N-type current through Ca V 2.2 channels was almost abolished when co-expressed transiently with g 7 in either Xenopus oocytes or COS-7 cells. Furthermore, immunocytochemistry and western blots show that g 7 has this effect by causing a large reduction in expression of Ca V 2.2 rather than by interfering with traf®ck-ing or biophysical properties of the channel. No effect of transiently expressed g 7 was observed on pre-existing endogenous N-type calcium channels in sympathetic neurones. Low homology to the stargazin-like g subunits, different gene structure and the unique functional properties of g 7 imply that it represents a distinct subdivision of the family of proteins identi®ed by their structural and sequence homology to stargazin.

Specific heterotrimeric G proteins composed of different ␣, ␤, and ␥ subunits transmit signals from membrane receptors to intracellular effectors (1-2). When a receptor is activated by an agonist, it catalyzes the exchange of GDP for GTP on the ␣ subunit of G proteins, resulting in dissociation of ␣ subunits from the ␤␥ dimers. It is now well documented that both the G␣ subunit and the G␤␥ complex are able to transmit signals to effector molecules (3-4). After the initial observation that G␤␥ could activate K ϩ channels (5), it was found that G␤␥ can regulate certain isoforms of adenylyl cyclase (6) and phospholipase C-␤ (7), activate the mitogen-activated protein kinase (8) and c-Jun N-terminal kinase (9) pathways, and mediate the translocation of the ␤-adrenergic receptor kinase (␤ARK) 1 (10). In portal vein myocytes, the G protein heterotrimer that couples angiotensin AT 1A receptors to increase [Ca 2ϩ ] i has been identified using an antisense oligonucleotide strategy. The G protein is composed of ␣ 13 , ␤ 1 , and ␥ 3 subunits, all three being required for activation of the transduction pathway (11 (14). The purpose of the present study was to identify which G protein subunits transduce the signal for activation of Ca 2ϩ channels after stimulation of the angiotensin AT 1 receptor in rat portal vein myocytes. A specific G␣ 13 function-blocking antibody and G␣ 13 peptide (corresponding to the last 11 amino acids of the carboxyl terminus) abrogated AII-induced stimulation of Ca 2ϩ channel current when dialyzed into the cells through the patch pipette. Intracellular infusion of specific G␤␥ binding agents, i.e. anti-␤ com antibody and ␤ARK peptide, also blocked the AII-induced stimulation of Ca 2ϩ channel current. Finally, overexpression of a ␤ARK 1 fragment and of G␤␥ scavengers, i.e. wild type of G␣ o1 and G␣ 12 subunits, largely inhibited the AII-induced increase in [Ca 2ϩ ] i . We conclude that the angiotensin AT 1 receptor uses the ␤␥ dimers of G 13 to transduce the signal leading to activation of Ca 2ϩ channels. EXPERIMENTAL PROCEDURESCell Preparation-Isolated myocytes from rat portal vein were obtained by enzymatic dispersion, as described previously (15). Cells were seeded at a density of about 10 3 cells/mm 2 on glass slides imprinted with squares for localization of injected cells and maintained in shortterm primary culture in M199 medium containing 2% fetal calf serum, 2 mM glutamine, 1 mM pyruvate, 200 units/ml penicillin, and 200 g/ml streptomycin; they were kept in an incubator gassed with 95% air, 5% CO 2 at 37°C and used within 72 h.Membrane current and [Ca 2ϩ ] i Measurements-Voltage clamp and membrane current recordings were made with a standard patch-clamp technique using a List EPC-7 patch-clamp amplifier (Darmstadt-Eberstadt, Germany). Whole-cell recordings were performed with patch pipettes having resistances of 1-3 megaohms. Membrane potential and current records were stored and analyzed using an IBM PC computer (P-clamp system, Axon Instruments, Inc., Foster City, CA). Simultane-